Despite significant advances in studies of the subclasses of opiate receptors and of their endogenous ligands, the molecular basis of opiate tolerance and dependence is not clearly understood. the long-term objective of the current proposal is to advance the understanding of the neurobiology of drug abuse which continues to be a major health problem in this country. Specifically this proposal focuses on the regulation of one endogenous opioid system proopiomelanocortin (POMC), in brain. Mechanisms by which POMC (the beta-endorphin (beta-EP) precursor) is regulated in brain by exogenous and endogenous opioids will be established in the rat by a coordinated study of POMC gene expression, precursor processing and peptide release. Effects of opioids on POMC mRNA levels in the hypothalamus will be determined by a sensitive solution hybridization assay. Studies will be performed in morphine tolerant rats and during morphine withdrawal to determine if POMC gene expression is down regulated in the morphine tolerant rat. POMC gene expression will also be studied in the presence of opiate receptor blockade or passive immunization with a specific beta-EP antiserum to determine if there is inhibitory feedback regulation of POMC by beta-EP. Opioid regulation of the posttranslational processing of POMC will be studied by HPLC and well characterized RIAs for the major POMC peptide products in brain. It is hypothesized that the processing of POMC may be an important regulator of beta-EP activity since the activity of beta-EP may be influenced by the simultaneous release of other peptides from the same precursor or by C-terminal processing of beta- EP itself. Opioid regulation of POMC peptide release will be studied in the perfused hypothalamus in vitro from animals that have been treated with opiate agonists and antagonists in vivo. Thus by a combination of techniques we hope to better assess beta-EP synthesis and turnover in brain and to re-examine the hypothesis that chronic opiate treatment might result in feedback inhibition of endogenous opioid peptide biosynthesis and release as one potential mechanism for opiate tolerance and dependence.